Chimeric Virus-Like Particles Incorporating Fusion GPI Anchored GM-CSF and IL-4 Conjugates

ABSTRACT

This disclosure relates to a GM-CSF and IL-4 conjugate fused to a glycolipid (GPI)-anchoring sequence that is incorporated into chimeric virus-like particles (VLPs) enriched with a viral protein, e.g., viral envelope protein or HIV envelope protein. In certain embodiments, the disclosure relates to methods of immunization with the chimeric VLPs disclosed herein. In certain embodiments, the disclosure relates to methods of immunization with disclosed HIV antigen containing VLPs through an intramuscular priming-intranasal boosting immunization route.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/154,256 filed Apr. 29, 2015. The entirety of this application ishereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant R01AI101047awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 14216US_ST25.txt. The text file is 8 KB, wascreated on Apr. 29, 2016, and is being submitted electronically viaEFS-Web.

BACKGROUND

Combination antiretroviral therapy (ART) has shown extraordinary successin reducing HIV transmission and prolonging life of subjects with HIV.However, in the vast majority of instances, ART does not entirely clearthe virus, and may people continue to become newly infected. Thus, thereis a need to identify an effective HIV vaccination method. Skountzou etal., report incorporation of glycosylphosphatidylinositol-anchoredgranulocyte-macrophage colony-stimulating factor or CD40 ligand enhancesimmunogenicity of chimeric simian immunodeficiency virus-like particles.J Virol, 2007, 81(3):1083-1094. See also Hellerstein et al. Hum VaccinImmunother. 2012, 8(11):1654-8.

Interleukin 4 (IL-4) serves as a signal to activate and elicit antibodyclass switching by B lymphocytes and converts naive helper T lymphocytesto active T lymphocytes. U.S. Pat. No. 6,838,081 reports enhancing thedevelopment of antigen presenting cells from precursor cells byadministering a combination of IL-4 and GM-CSF. See also, U.S. PatentApplication 2004/0072299, and Hikino et al., Anticancer Res, 24:1609-1616 (2004). GIFT fusokines are the fused proteins derived fromgranulocyte-macrophage colony-stimulating factor (GM-CSF) and cytokinetransgenes. Deng et al. report a fusokine, GIFT4, generated byN-terminal coupling of GM-CSF to interleukin-4 (IL4). Cancer Res, 2014,74:4133-4144. See also WO2014/066443.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to a GM-CSF and IL-4 conjugate fused to aglycolipid (GPI)-anchoring sequence that is incorporated into chimericvirus-like particles (VLPs) enriched with a viral protein, e.g., viralenvelope protein or HIV envelope protein. In certain embodiments, thedisclosure relates to methods of immunization with the chimeric VLPsdisclosed herein. In certain embodiments, the disclosure relates tomethods of immunization with disclosed HIV antigen containing VLPsthrough an intramuscular priming-intranasal boosting immunization route.

In certain embodiments, the disclosure relates to nucleic acidscomprising a segment encoding a fusion protein comprisinggranulocyte-macrophage colony-stimulating factor and interleukin 4 and aglycosylphosphatidylinositol signal sequence. In certain embodiments,the granulocyte-macrophage colony-stimulating factor segment comprisesMWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO: 1) or variants thereof with greaterthan 70% identity. In certain embodiments, the interleukin 4 segmentcomprises MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS (SEQ ID NO: 2) or variants thereofwith greater than 70% identity. In certain embodiments, theglycosylphosphatidylinositol signal sequence CD59 segment comprisesLSNGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 5) or variants thereof withgreater than 70% identity. Other glycosylphosphatidylinositol signalsequences are contemplated such as, the GPI anchor sequence of humanLFA3, CD55, human Fcγ receptor III (CD16b). See Kueng et al., J Virol,2007, 81(16):8666-8676.

In certain embodiments, the fusion protein further comprises a melittinsignal peptide segment comprising MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 6)or variants thereof with greater than 70% identity. In certainembodiments, the fusion protein segment comprises or consistsessentially ofMKFLVNVALVFMVVYISYIYAMWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQEGGGGSMGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSSLSNGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 7), orvariants thereof with greater than 70% identity.

In certain embodiments, the disclosure relates to chimeric virus-likeparticle comprising the fusion protein disclosed herein. In certainembodiments, the disclosure relates to isolated fusion proteins encodeddisclosed herein. In certain embodiments, the disclosure relates tovectors comprising the nucleic acids arranged as disclosed herein inoperable combination with a promoter sequence. In certain embodiments,the chimeric virus-like particle further comprises a viral protein orenvelope protein, e.g., HIV gp160, gp120, or gp41 or an influenza,hemagglutinin, HA1, A, B, C, and E antigenic epitopes on the HA1 subunitof the influenza virus hemagglutinin, neuraminidase, M2 protein, M2eprotein, M1 protein, or combinations thereof.

In certain embodiments, the disclosure relates to methods of vaccinationcomprising administering an effective amount of the chimeric virus-likeparticle disclosed herein to a subject in need thereof. In certainembodiments, the disclosure relates to methods of treating or preventinga viral infection comprising administering an effective amount of thechimeric virus-like particle as disclosed herein to a subject in needthereof. In certain embodiments, the administration is intramuscular. Incertain embodiments, a second administration is provided more than twoweeks after an initial administration. In certain embodiments, thesecond administration is intranasal. In certain embodiments, the methodsfurther comprise a third administration. In certain embodiments, themethods further comprise administration is in combination with theadministration of another antiviral agent, antigen, adjuvant, orvaccine.

In certain embodiments, the disclosure relates to pharmaceutical orvaccine compositions comprising a chimeric virus-like particlecomprising a GM-CSF and IL-4 conjugate fused to a glycolipid(GPI)-anchoring sequence that is incorporated into chimeric virus-likeparticles (VLPs) and alternative embodiments disclosed herein and apharmaceutically acceptable excipient.

In certain embodiments, a vaccine composition comprises a chimericvirus-like particle comprising GM-CSF and IL-4 conjugate fused to aglycolipid (GPI)-anchoring sequence that is incorporated into chimericvirus-like particles (VLPs) and an antigen, optionally additional anadjuvant.

In certain embodiments, this disclosure relates to conjugates comprisinga GM-CSF and IL-4 conjugate fused to a glycolipid (GPI)-anchoringsequence connected by a linker, e.g., polypeptide. In certainembodiments, the disclosure relates to isolated nucleic acids encodingthese polypeptide conjugates, vectors comprising nucleic acid encodingthe fusion proteins, and protein expression systems comprising thesevectors, e.g., recombinant infectious viral particles and host cellscomprising such nucleic acids.

In some embodiments, the vaccine comprises, or is the antigeniccomponent of, a live attenuated virus, killed virus, a virus-likeparticle, virosome, and the antigen is typically a viral protein orglycoprotein.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection comprising administering an effectiveamount of a pharmaceutical composition comprising a chimeric virus-likeparticle comprising GM-CSF and IL-4 conjugate fused to a glycolipid(GPI)-anchoring sequence optionally in combination with a vaccine orantigen and optionally an adjuvant. In certain embodiments, the subjectis at risk or, exhibiting symptoms of, or diagnosed with a viralinfection, such as a chronic viral infection.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection comprising administering an effectiveamount of a vaccine comprising a chimeric virus- like particlecomprising GM-CSF and IL-4 conjugate fused to a glycolipid(GPI)-anchoring sequence wherein the subject is diagnosed with influenzaA virus including subtype H1N1, influenza B virus, influenza C virus,rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARScoronavirus, human adenovirus types (HAdV-1 to 55), human papillomavirus(HPV) Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59,parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus,Merkel cell polyomavirus, coxsackie A virus, norovirus, Rubella virus,lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measlesvirus, mumps virus, respiratory syncytial virus, rinderpest virus,California encephalitis virus, hantavirus, rabies virus, ebola virus,marburg virus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2(HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV),cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, orKaposi's sarcoma-associated herpesvirus, hepatitis A, hepatitis B,hepatitis C, hepatitis D, hepatitis E, or human immunodeficiency virus(HIV).

In certain embodiments, the disclosure relates to administering achimeric virus-like particle comprising GM-CSF and IL-4 conjugate fusedto a glycolipid (GPI)-anchoring sequence in combination with anantiviral agent such as abacavir, acyclovir, acyclovir, adefovir,amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla,boceprevir, cidofovir, combivir,darunavir, delavirdine, didanosine,docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a,penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir,ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine,tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir,tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc,vidarabine, viramidine zalcitabine, zanamivir, and/or zidovudine.

In certain embodiments, the disclosure relates to gene therapiescomprising administering vectors comprising nucleic acid encodingconjugates disclosed herein to a subject in need thereof. In certainembodiments, the nucleic acids are isolated and/or purified from theirnatural state or translated to a non-naturally occurring form such ascDNA.

In certain embodiments, it is contemplated that chimeric virus-likeparticles disclosed herein may contain other surface polypeptides,antigens and co-stimulatory molecules such as B7-1, B7-2, ICAM-1, and/orIL-2. It is contemplated that these particles may be used in all theapplications that chimeric virus-like particles disclosed herein arementioned.

Within certain embodiments, GM-CSF and IL-4 conjugates fused to aglycolipid (GPI)-anchoring sequence may be further conjugated to anadjuvant, cytokine, co-stimulatory molecule, antigen, protein, orglycoprotein. In certain embodiments, the antigen is a viral protein.

In certain embodiments, the viral protein or antigen is selected from aninfluenza virus hemagglutinin and neuraminidase; cytomegalovirusglycoprotein gB, p28, p38, p50, p52, p65, and p150; Borrelia p41; HIVnef, integrase, gag, protease, tat, env, p31, p17, p24, p31, p55, p66,gp32, gp36, gp39, gp41, gp120, and gp160; SIV p55; HBV core, surfaceantigen, and australian antigen; HCV core nucleocapsid, NS3, NS4, andNS5; Dengue env and NS1; EBV early antigen, p18, p23, gp125, nuclearantigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein(EBNA-LP), latent membrane proteins (LMP)-1, LMP-2A and LMP-2B; andherpes simplex virus gD and gG or fragments or mutated forms thereof.

In certain embodiments, the adjuvant or cytokine is selected from IL-2,IL-12, IL-15, IL-7, IL-18, IL-21, IL-27, IL-31, IFN-alpha, flagellin,unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, andheat stable antigen (HSA).

In certain embodiments, the disclosure contemplates administration ofpharmaceutical products comprising chimeric virus-like particlesdisclosed herein by intranasal (IN), intravenous (IV), subcutaneous(SC), or intraperitoneal (IP) administration.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates the GPI-GIFT4 encoding gene. Coding sequences forthe melittin signal peptide and murine CD59-GPI anchor were fused at the5′- and 3′-ends of GIFT4 encoding DNA, respectively, to form thefull-length gene.

FIG. 1B shows data on the GPI-GIFT4 cellular expression. Western blotswere developed with anti-GM-CSF antibody. Lane 1, whole cell lysate ofGPI-GIFT4-expressing rBV infected insect cell sample; Lane 2, purifiedGM-CSF protein; Lane 3, whole cell lysate of HIV Env-expressing rBVinfected cells as a control.

FIG. 2 shows western blotting analysis of the protein components ofVLPs. VLP samples containing 1 μg of total protein were loaded forSDS-PAGE followed by western blotting. The Env incorporated intostandard Env/Gag VLPs (sVLPs, lane 2) or cVLPs (lane 4) was observed tohave a molecular mass of about 120 kDa. Protein bands were probed with:a.1, anti-HIV Gag antibody; a.2, anti-gp120 polyclonal antibodies; a.3,anti-GM-CSF antibody. Lane 1, Gag only VLPs; Lane 2, sVLPs; Lane 3,GIFT4/Gag VLPs; lane 4, cVLPs; M, Molecular weight (kDs).

FIG. 3A shows data on serum endpoint titers. Guinea pigs were immunizedwith Gag only VLPs, GIFT4/Gag VLPs, sVLPs, or cVLPs (Left to Right) inthe one i.m. priming-two i.n. boosting route at weeks 0, 4, 8,respectively, and immune sere were collected 2 weeks after eachimmunization at weeks 2, 6, 10, respectively.

FIG. 3B shows data for IgG1 titers of immune sera from the bleed (week10).

FIG. 3C shows data for IgG2 endpoint titers of immune sera from thebleed (week 10) as in FIG. 3A.

FIG. 4A shows data on mucosal antibody endpoint titers (saliva IgG).Mucosal samples were collected at week 12, 4 weeks after the lastboosting immunization. Env-specific IgG and IgA endpoint titers weredetected by ELISA as described in Materials and Methods.

FIG. 4B shows data for saliva IgA as in FIG. 4A.

FIG. 4C shows data for vaginal IgG as in FIG. 4A.

FIG. 4D shows data for vaginal IgA as in FIG. 4A.

FIG. 5A shows avidity indexes of immune serum IgG to clade Bpseudoviruses Avidity assays were conducted with immune sera of bleed 3(at week 10) from sVLP (Right) and cVLPs (Left)-immunized guinea pigs.

FIG. 5B shows avidity indexes of immune serum IgG to clade Cpseudoviruses as in FIG. 5A.

FIG. 6A shows data on neutralizing activity against clade Bpseudoviruses. Immune sera of the bleed 3 from sVLPs (Right) and cVLPs(Left)-immunized animals were tested for neutralizing activity. Thefinal dilution factor of immune sera was 40-fold.

FIG. 6B shows data on neutralization against clade C pseudoviruses as inFIG. 6A.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

As used herein, the term “conjugate” refers to molecular entities joinedby covalent bonds or other arrangement that provides substantiallyirreversible binding under physiological conditions. For example, twoproteins, isolated and/or purified polypeptide sequence, may beconjugated together by a linker polymer, e.g., amino acid, polypeptidesequence, ethylene glycol polymer. Two proteins may be conjugatedtogether by linking one protein to a ligand and linking the secondprotein to a receptor, e.g., streptavidin and biotin or an antibody andan epitope.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “subject” refers to any animal, typically a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, “amino acid sequence” refers to an amino acid sequenceof a protein molecule. An “amino acid sequence” can be deduced from thenucleic acid sequence encoding the protein. However, terms such as“polypeptide” or “protein” are not meant to limit the amino acidsequence to the deduced amino acid sequence, but include non-naturallyoccurring amino acids, post-translational modifications of the deducedamino acid sequences, such as amino acid deletions, additions, andmodifications such as glycosylation and addition of lipid moieties.

The term “a nucleic acid sequence encoding” a specified polypeptiderefers to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in a cDNA, genomic DNA or RNAform. When present in a DNA form, the oligonucleotide, polynucleotide,or nucleic acid may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present disclosure may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

A “virus-like particle” refers to a particle comprising virion proteinsbut is substantially free of viral genetic material, e.g., viral RNA.Virus-like particles may contain viral proteins from different viruses.See e.g., Guo et al., Enhancement of mucosal immune responses bychimeric influenza HA/SHIV virus-like particles, Virology, 2003,313(2):502-13. Virus-like particles may contain lipid membranes and maybe constructed to express a variety of antigens on their particlesurface ether by expression in viral vectors use to create the particlesor by mixing the virus-like particle with an antigen or otherpolypeptide conjugated to a glycosylphosphatidyl-inositol anchor. Seee.g. Skountzou et al., J. Virol., 2007, 81(3):1083-94; Derdak et al.,PNAS, 2006, 103(35) 13144-13149; Poloso et al., Molecular Immunol, 2001,38:803-816.

Incorporation of a GPI-Anchored Engineered Cytokine as a MolecularAdjuvant Enhances the Immunogenicity of HIV VLPs

Wang et al. report incorporation of chimeric human immunodeficiencyvirus envelope glycoproteins into virus-like particles. J Virol, 2007,81, 10869-10878. A fusokine (fusion protein from two cytokines) fromGM-CSF and IL4 (GIFT4) leads to B-cell effector function manifest by analtered pro-immune cytokine secretory profile and a B-cell mitogenicresponse. See Deng, et al. Cancer Res, 74, 4133-4144 (2014). Amembrane-bound form of GIFT4 was generated by fusing the CD59 glycolipid(glycosylphosphatidyl-inositol, GPI) signal sequence to the GIFT4C-terminal sequence in frame, and incorporated the membrane-anchoredGIFT4 into Env-enriched VLPs. These chimeric VLPs (cVLPs) harboring botha high density of Env and membrane-anchored GIFT4 elicited highlyaugmented Env-specific antibody responses with improved quality, asreflected by enhanced avidity and neutralization activity.

A goal of developing vaccines against HIV is to elicit immune responsesat the level of the mucosal surface to block transmission, as well as insystemic compartments to clear disseminated viruses. Vaccinesadministered by systemic routes generally fail to stimulate strongmucosal immune responses. Chimeric VLPs were developed with efficientco-incorporation of HIV Env together with a fusokine, GIFT4, as aco-stimulatory factor, into the same particles. The resulting cVLPs wereevaluated for their capacity to elicit enhanced antibody responses insystemic compartments as well as mucosal surfaces. This disclosureintegrates several approaches to enhance HIV immune responses. Theseinclude: 1) enhanced incorporation of Env into VLPs to increase thedensity of immunogens; 2) construction and co-incorporation of amembrane-bound form of GIFT4 into cVLPs to further improveimmunogenicity by stimulating B lymphocyte proliferation and activation;3) employment of a systemic prime/mucosal boost route to inducedsystemic as well as mucosal immune responses.

Vaccines are typically administered by intramuscular injection, andinduce systemic immune responses. However, mucosal immunity is rarelyinduced by systemic immunization. Since mucosal transmission is thepredominant pathway for HIV infection and accounts for as high as 80% ofAIDS incidence globally, immunity functioning at the mucosal portals ofentry is important for preventing primary HIV-1 infection. Because ofthe relatively low efficiency of HIV infection at mucosal surfaces, evena modest enhancement of antibody-mediated protective mucosal immuneresponses could have a significant effect on reducing disease incidence.Thus an effective HIV vaccine may need to induce both mucosal immunityto reduce the frequency of initial infection and possibly block theescape of virus from the genital and intestinal mucosa into systemiclymphoid organs, and systemic immunity, such as broadly neutralizingantibody responses, to clear any disseminated virus.

An intramuscular prime-intranasal boost immunization route was employedto induced enhance immunity both at systemic compartments and mucosalsurfaces. Germinal centers containing B, T, plasma, and professionalantigen presenting cells (APCs) are present in the nasal cavity.Lymphoid tissues strategically positioned at the site of entry of therespiratory and the digestive tracts are important in antigen uptake. Bytwo i.n. boosts with the constructed cVLPs, highly elevated mucosal HIVEnv-specific IgG and IgA levels were induced compared to sVLPsadministered by the same route. The antibodies secreted into the lumenprovide an immunological barrier to limit the penetration of antigensinto mucous membranes and are associated with protection from HIV-1infection. Although mucosal IgA and IgG responses were observed inanimals immunized with cVLPs, serum IgA was not induced.

In a recent analysis of immune correlates in the RV144 trial, serum IgAlevels were found to be inversely correlated to HIV protection. SeeHaynes et al. Immune-correlates analysis of an HIV-1 vaccine efficacytrial. NEJM, 2012, 366, 1275-1286. Low serum IgA levels in immune seramay therefore be an advantageous consequence of GIFT4-containingVLP-induced immunity.

A desirable antibody response should include high titers of antibodieswith high antibody avidity and neutralizing activity. Enhanced antibodyavidity and neutralizing breadth and potency in the cVLP-immunized groupwas observed, supporting the conclusion that the adjuvant effect ofGPI-GIFT4 enables the immune system to more effectively process orpresent B cell epitopes residing in Env. The enhanced avidity observedmay be contributed by non-neutralizing antibodies.

GM-CSF and IL-4 Conjugate (GIFT4) Fused to a Glycolipid (GPI)-AnchoringSequence (GPI-GIFT4) that is Incorporated into Chimeric Virus-LikeParticles (cVLPs) Enriched with a Viral Antigen

Some exemplary methods of making GM-CSF and IL-4 conjugate fused to aglycolipid (GPI)-anchoring sequence that is incorporated into chimericvirus-like particles (cVLPs) are described below. Methods useful for themaking VLPs of the present disclosure for administration with vaccines,viral proteins, antigens and adjuvant molecules of the presentdisclosure, or which incorporate viral proteins, antigen, or adjuvantmolecules into the VLP, may be found in U.S. Pat. Nos. 8,795,682;6,077,662; WO 2004/042001, which are herein incorporated by reference intheir entireties, and which are also described below.

VLPs for use in the immunogenic compositions of the present disclosurecan be produced by in vitro cell culture expression systems such as, butnot limited to, recombinant baculovirus expression system (BEVS) (see,for example, Yamshchikov et al., (1995) Virology: 214, 50-58). Assemblyof HIV or SIV virus-like particles containing envelope proteins may beperformed using expression systems, such as, but not limited to, abaculovirus expression system (Yamshchikov et al., (1995) Virology: 214,50-58), recombinant poxvirus expression system (MVA) (Wyatt et al.,(1996), Vaccine: 15, 1451-1458), recombinant VSV, recombinantadenovirus, and recombinant DNA expression vectors. Preferably, the VLPsare produced using recombinant BEVS and recombinant poxvirus expressionsystems.

In general, VLPs can be produced by simultaneously introducing into acell a viral core protein expression vector, a viral surface envelopeglycoprotein expression vector, and/or an expression vector encodingGPI-GIFT4. The expressed viral core protein self-assembles into a VLPthat incorporates the viral surface envelope glycoprotein and/or theGPI-GIFT4. The viral surface envelope glycoprotein and/or the adjuvantmolecule are expressed and disposed on the VLP surface. Thereafter, thecell produces the VLP (for example, Vero cells, chimeric and/orphenotypically mixed VLPs). The cells may be selected from, but are notlimited to, insect cells (e.g., Spodopera frugiperda Sf9 and Sf21cells), and mammalian cells such as, but not limited to, EL4 cells andHeLa cells. The expression elements for expressing the viral coreprotein, viral surface envelope glycoprotein, and GPI-GIFT4 can also beincluded together in a single expression vector, or can be included intwo or more expression vectors.

In general, the viral protein expression vector can be produced byoperably linking a coding sequence for a viral protein of a virus to anappropriate promoter (e.g., an early promoter, late promoter, or hybridlate/very late promoter). The viral protein expression vector can alsobe modified to form a viral protein expression construct. In addition,the viral surface envelope glycoprotein expression vector can beproduced by operably linking a coding sequence for a viral surfaceenvelope glycoprotein of a virus to an appropriate promoter (e.g., earlypromoter, late promoter, or hybrid late/very late promoter). The viralsurface envelope glycoprotein expression vector can be modified to forma viral surface envelope glycoprotein expression construct. Similarly,the GPI-GIFT4 expression vector can be produced by operably linking acoding sequence for GPI-GIFT4 to an appropriate promoter (e.g., earlypromoter, late promoter, or hybrid late/very late promoter). TheGPI-GIFT4 expression vector can be modified to form an GPI-GIFT4expression construct.

In other embodiments of the disclosure, nucleic acid sequences encodingfor a viral core protein, at least one viral surface envelope protein,and GPI-GIFT4 can be included in a single expression vector, or in twoor more expression vectors. The one or more expression vectors can beintroduced into a host cell, the proteins can be expressed in the cell,whereby the cell forms the VLP. In embodiments, each of the nucleic acidsequences encoding for the viral core protein, the viral surfaceenvelope glycoprotein, and the GPI-GIFT4 is operably linked to anappropriate promoter (e.g., a baculovirus promoter, a recombinantModified Vaccinia Ankara (MVA) promoter, a CMV promoter, an EF promoter,an adenovirus promoter, a recombinant VSV promoter, a recombinantadenovirus promoter, a recombinant alphavirus promoter, and arecombinant DNA expression vector). Appropriate promoters include, butare not limited to, a constitutive or inducible promoter; an early,late, or hybrid late/very late promoter.

An embodiment of this disclosure, GPI-GIFT4 comprises a human GM-CSF,MWLQSLLLLGTV ACSISAPARS PSPSTQPWEHVNAI QEARRLLN LSRDTAAEMN ETVEVISEMFDLQEPTC LQTRLELYKQGL RGSLTKLKGPLTMMASH YKQHCPPETSCATQ TITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO: 1) sequence and a human IL-4. Incertain embodiments, the disclosure contemplates a fusokine with arecombinant human form such as isoform 1 which is amino acids sequenceMGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAASKNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGLNSCPVKEANQ STLENFLERL KTIMREKYSK CSS (SEQ ID NO: 2) or isoform 2 whichis amino acid sequence MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNSLTEQKNTTEK ETFCRAATVL RQFYSHHEKD TRCLGATAQQ FHRHKQLIRF LKRLDRNLWGLAGLNSCPVK EANQSTLENFLERLKTIMRE KYSKCSS (SEQ ID NO: 3). Isoform 2 lacksan in-frame exon in the 5′ region, compared to variant 1, resulting anisoform (2) that lacks an internal region, as compared to isoform 1.

The present disclosure encompasses fusion proteins involving full-lengthpre-processed forms, as well as mature processed forms, fragmentsthereof and variants of each or both of the GM-C SF and IL-4 entitieswith linker amino acids, including allelic as well as non-naturallyoccurring variants. In addition to naturally-occurring allelic variantsof the GM-CSF and IL-4 entities that may exist in the population, theskilled artisan will further appreciate that changes (i.e. one or moredeletions, additions and/or substitutions of one or more amino acid) canbe introduced by mutation using classic or recombinant techniques toeffect random or targeted mutagenesis. A suitable variant in use in thepresent disclosure typically has an amino acid sequence having a highdegree of homology with the amino acid sequence of the correspondingnative cytokine. In one embodiment, the amino acid sequence of thevariant cytokine in use in the fusion protein of the disclosure is atleast 70%, at least about 75%, at least about 80%, at least about 90%,typically at least about 95%, more typically at least about 97% and evenmore typically at least about 99% identical to the corresponding nativesequence, e.g., SEQ ID NO: 7. In certain embodiments, such nativesequence is of human GM-CSF and/or human IL-4.

Percent identities between amino acid or nucleic acid sequences can bedetermined using standard methods known to those of skill in the art.For instance for determining the percentage of homology between twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes. The amino acid residues at corresponding amino acid positionsare then compared. Gaps can be introduced in one or both amino acidsequence(s) for optimal alignment and non- homologous sequences can bedisregarded for comparison purposes. When a position in the firstsequence is occupied by the same amino acid residue as the correspondingposition in the second sequence, then the sequences are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps which need to be introduced foroptimal alignment and the length of each gap. The comparison ofsequences and determination of percent identity and similarity betweentwo sequences can be accomplished using a mathematical algorithm (e.g.Computational Molecular Biology, 1988, Ed Lesk A M, Oxford UniversityPress, New York; Biocomputing: Informatics and Genome Projects, 1993, EdSmith D. W., Academic Press, New York; Computer Analysis of SequenceData, 1994, Eds Griffin A. M. and Griffin H. G., Human Press, NewJersey; Sequence Analysis Primer, 1991, Eds Griskov M. and Devereux J.,Stockton Press, New York). Moreover, various computer programs areavailable to determine percentage identities between amino acidsequences and between nucleic acid sequences, such as GCG™ program(available from Genetics Computer Group, Madison, Wis.), DNAsis™ program(available from Hitachi Software, San Bruno, Calif.) or the MacVector™program (available from the Eastman Kodak Company, New Haven, Conn.).

Suitable variants of GM-CSF and IL-4 entities for use in the presentdisclosure are biologically active and retain at least one of theactivities described herein in connection with the correspondingpolypeptide. Typically, the therapeutic effect is preserved, although agiven function of the polypeptide(s) may be positively or negativelyaffected to some degree, e.g. with variants exhibiting reducedcytotoxicity or enhanced biological activity. Amino acids that areessential for a given function can be identified by methods known in theart, such as by site-directed mutagenesis. Amino acids that are criticalfor binding a partner/substrate (e.g. a receptor) can also be determinedby structural analysis such as crystallization, nuclear magneticresonance and/or photoaffinity labeling. The resulting variant can betested for biological activity in assays such as those described above.

For example, in one class of functional variants, one or more amino acidresidues are conservatively substituted. A “conservative amino acidsubstitution” is one in which the amino acid residue in the nativepolypeptide is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. Typically, substitutions are regarded asconservative when the replacement, one for another, is among thealiphatic amino acids Ala, Val, Leu, and Ile; the hydroxyl residues Serand Thr; the acidic residues Asp and Glu; the amide residues Asn andGln; the basic residues Lys and Arg; or the aromatic residues Phe andTyr. Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a cytokine coding sequence, such as bysaturation mutagenesis, and the resultant mutant can be screened for itsbiological activity as described herein to identify mutants that retainat least therapeutic activity.

Although the GM-C SF and IL-4 entities can be directly fused in thefusion protein of the disclosure, it is however typical to use a linkerfor joining GM-CSF and IL-4. The purpose of the linker is to allow thecorrect formation, folding and/or functioning of each of the GM-CSF andIL-4 entities. It should be sufficiently flexible and sufficiently longto achieve that purpose. Typically, the coding sequence of the linkermay be chosen such that it encourages translational pausing andtherefore independent folding of the GM-CSF and IL-4 entities. A personskilled in the art will be able to design suitable linkers in accordancewith the disclosure. Certain embodiments of the disclosure are notlimited by the form, size or number of linker sequences employed.Multiple copies of the linker sequence of choice may be inserted betweenGM-CSF and IL-4. The only requirement for the linker sequence is that itfunctionally does not adversely interfere with the folding and/orfunctioning of the individual entities of the fusion protein. Forexample, a suitable linker is 1 to 5 or 5 to 50 amino acid long and maycomprise amino acids such as glycine, serine, threonine, asparagine,alanine and proline (see for example Wiederrecht et al., 1988, Cell 54,841; Dekker et al., 1993, Nature 362, 852; Sturm et al., 1988, Genes andDev. 2, 1582; Aumailly et al., 1990 FEBS Lett. 262, 82). Repeatscomprising serine and glycine residues are typical in the context of thedisclosure. Specific examples of suitable linkers consists of two orthree or more (e.g. up to eight or more) copies of the sequenceGly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 4). It will be evident that thedisclosure is not limited to the use of these particular linkers.

The disclosure further includes fusion proteins which comprise, oralternatively consist essentially of, or alternatively consist of anamino acid sequence which is at least 70%, 75%, 80%, 90%, 95%, 97%, 99%homologous or even better 100% homologous (identical) to all or part ofany of the amino acid sequences recited in SEQ ID NO: 1-7.

In the context of the present disclosure, a protein “consists of” anamino acid sequence when the protein does not contain any amino acidsbut the recited amino acid sequence. A protein “consists essentially of”an amino acid sequence when such an amino acid sequence is presenttogether with only a few additional amino acid residues, typically fromabout 1 to about 50 or so additional residues. A protein “comprises” anamino acid sequence when the amino acid sequence is at least part of thefinal (i.e. mature) amino acid sequence of the protein. Such a proteincan have a few up to several hundred additional amino acids residues.Such additional amino acid residues can be naturally associated witheach or both entities contained in the fusion or heterologous aminoacid/peptide sequences (heterologous with respect to the respectiveentities). Such additional amino acid residues may play a role inprocessing of the fusion protein from a precursor to a mature form, mayfacilitate protein trafficking, prolong or shorten protein half-life orfacilitate manipulation of the fusion protein for assay or production,among other things. Typically, the fusion proteins of the disclosurecomprise a signal peptide at the NH₂-terminus in order to promotesecretion in the host cell or organism. For example, the endogenoussignal peptide (i.e. naturally present in the cytokine present at theNH₂ terminus of said fusion) can be used or alternatively a suitableheterologous (with respect to the cytokine in question) signal peptidesequence can be added to the cytokine entity present at the NH₂ terminusof the fusion or inserted in replacement of the endogenous one.

In the context of the disclosure, the fusion proteins of the disclosurecan comprise cytokine entities of any origin, i.e. any human or animalsource (including canine, avian, bovine, murine, ovine, feline, porcine,etc). Although “chimeric” fusion proteins are also encompassed by thedisclosure (e.g. one cytokine entity of human origin and the other of ananimal source), it is typical that each entity be of the same origin(e.g. both from humans).

The fusion proteins of the present disclosure can be produced bystandard techniques. Polypeptide and DNA sequences for each of thecytokines involved in the fusion protein of the present disclosure arepublished in the art, as are methods for obtaining expression thereofthrough recombinant or chemical synthetic techniques. In anotherembodiment, a fusion-encoding DNA sequence can be synthesized byconventional techniques including automated DNA synthesizers. Then, theDNA sequence encoding the fusion protein may be constructed in a vectorand operably linked to a regulatory region capable of controllingexpression of the fusion protein in a host cell or organism. Techniquesfor cloning DNA sequences for instance in viral vectors or plasmids areknown to those of skill in the art (Sambrook et al, 2001, “MolecularCloning. A Laboratory Manual”, Laboratory Press, Cold Spring HarborN.Y.). The fusion protein of the disclosure can be purified from cellsthat have been transformed to express it.

The present disclosure also provides a nucleic acid molecule encodingthe fusion protein of the disclosure. Within the context of the presentdisclosure, the term “nucleic acid” and “polynucleotide” are usedinterchangeably and define a polymer of nucleotides of any length,either deoxyribonucleotide (DNA) molecules (e.g., cDNA or genomic DNA)and ribonucleotide (RNA) molecules (e.g., mRNA) and analogs of the DNAor RNA generated using nucleotide analogs (see U.S. Pat. No. 5,525,711and U.S. Pat. No. 4,711,955 as examples of nucleotide analogs). Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides mayalso be interrupted by non-nucleotide elements. The nucleic acidmolecule may be further modified after polymerization, such as byconjugation with a labeling component. The nucleic acid, especially DNA,can be double-stranded or single-stranded, but typically isdouble-stranded DNA. Single-stranded nucleic acids can be the codingstrand (sense strand) or the non-coding strand (anti-sense strand).

The nucleic acid molecules of the disclosure include, but are notlimited to, the sequence encoding the fusion protein alone, but maycomprise additional non-coding sequences, for example introns andnon-coding 5′ and 3′ sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and mRNA stability. For example, the nucleic acid molecule ofthe disclosure can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank (i.e.sequences located at the 5′ and 3′ ends) or are present within thegenomic DNA encoding GM-CSF and IL-4 entities.

According to a typical embodiment, the present disclosure providesnucleic acid molecules which comprise, or alternatively consistessentially of, or alternatively consist of a nucleotide sequenceencoding all or part of an amino acid sequence encoding a fusion proteinwhich is at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95%, typically at least about 97%, moretypically at least about 99% homologous or even more typically 100%homologous to any of the amino acid sequences shown in SEQ ID NO: 1-7.

In another embodiment, a nucleic acid molecule of the disclosurecomprises a nucleic acid molecule which is a complement of all or partof a nucleotide sequence encoding the fusion protein shown in any of SEQID NO: 1-7. A nucleic acid molecule which is complementary to thenucleotide sequence of the present disclosure is one which issufficiently complementary such that it can hybridize to thefusion-encoding nucleotide sequence under stringent conditions, therebyforming a stable duplex. Such stringent conditions are known to thoseskilled in the art. A typical, non-limiting example of stringenthybridization conditions are hybridization in 6 times sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2 times SSC, 0.1% SDS at 50-65 C. In one embodiment, thedisclosure pertains to antisense nucleic acid to the nucleic acidmolecules of the disclosure. The antisense nucleic acid can becomplementary to an entire coding strand, or to only a portion thereof.

In still another embodiment, the disclosure encompasses variants of theabove-described nucleic acid molecules of the disclosure e.g., thatencode variants of the fusion proteins that are described above. Thevariation(s) encompassed by the present disclosure can be created byintroducing one or more nucleotide substitutions, additions and/ordeletions into the nucleotide sequence by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Followingmutagenesis, the variant nucleic acid molecule can be expressedrecombinantly as described herein and the activity of the resultingprotein can be determined using, for example, assays described herein.Alternatively, the nucleic acid molecule of the disclosure can bealtered to provide preferential codon usage for a specific host cell(for example E. coli; Wada et al., 1992, Nucleic Acids Res. 20,2111-2118). The disclosure further encompasses nucleic acid moleculesthat differ due to the degeneracy of the genetic code and thus encodefor example the same fusion protein as any of those shown in SEQ ID NO:1-7.

Another embodiment of the disclosure pertains to fragments of thenucleic acid molecule of the disclosure, e.g. restriction endonucleaseand PCR-generated fragments. Such fragments can be used as probes,primers or fragments encoding an immunogenic portion of the fusionprotein.

The nucleic acid molecules of the present disclosure can be generatedusing the sequence information provided herein. The nucleic acidencoding each of the GM-CSF and IL-4 entities can be cloned or amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateprobes or oligonucleotide primers according to standard molecularbiology techniques (e.g., as described in Sambrook, et al. “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y., 2001) or standard PCRamplification techniques based on sequence data accessible in the art(such as those provided above in connection with the fusion proteins ofthe disclosure or those provided in the Examples part). Fusing of theGM-CSF sequence to the IL-4 sequence may be accomplished as described inthe Experimental below or by conventional techniques. For example, theGM-CSF and IL-4 encoding sequences can be ligated together in-frameeither directly or through a sequence encoding a peptide linker. TheGM-CSF-encoding sequence can also be inserted directly into a vectorwhich contains the IL-4-encoding sequence, or vice versa. Alternatively,PCR amplification of the GM-C SF and IL-4-encoding sequences can becarried out using primers which give rise to complementary overhangswhich can subsequently be annealed and re-amplified to generate a fusiongene sequence.

Pharmaceutical Compositions

As used herein the language “pharmaceutically acceptable excipient” isintended to include any and all carriers, solvents, diluents,excipients, adjuvants, dispersion media, coatings, antibacterial andantifungal agents, and absorption delaying agents, and the like,compatible with pharmaceutical administration.

Suitably, the pharmaceutical composition of the disclosure comprises acarrier and/or diluent appropriate for its delivering by injection to ahuman or animal organism. Such carrier and/or diluent is non-toxic atthe dosage and concentration employed. It is selected from those usuallyemployed to formulate compositions for parental administration in eitherunit dosage or multi-dose form or for direct infusion by continuous orperiodic infusion. It is typically isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as provided bysugars, polyalcohols and isotonic saline solutions. Representativeexamples include sterile water, physiological saline (e.g. sodiumchloride), bacteriostatic water, Ringer's solution, glucose orsaccharose solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions (see for example the most current edition ofRemington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott,Williams & Wilkins). The pH of the composition of the disclosure issuitably adjusted and buffered in order to be appropriate for use inhumans or animals, typically at a physiological or slightly basic pH(between about pH 8 to about pH 9, with a special preference for pH8.5). Suitable buffers include phosphate buffer (e.g. PBS), bicarbonatebuffer and/or Tris buffer. A typical composition is formulated in 1Msaccharose, 150 mM NaCl, 1 mM MgCl2, 54 mg/1 Tween 80, 10 mM Tris pH8.5. Another typical composition is formulated in 10 mg/ml mannitol, 1mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.

The composition of the disclosure can be in various forms, e.g. in solid(e.g. powder, lyophilized form), or liquid (e.g. aqueous). In the caseof solid compositions, the typical methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof. Such solutions can, if desired, be stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection.

Nebulized or aerosolized formulations also form part of this disclosure.Methods of intranasal administration are well known in the art,including the administration of a droplet, spray, or dry powdered formof the composition into the nasopharynx of the individual to be treatedfrom a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer (see forexample WO 95/11664). Enteric formulations such as gastroresistantcapsules and granules for oral administration, suppositories for rectalor vaginal administration also form part of this disclosure. Fornon-parental administration, the compositions can also includeabsorption enhancers which increase the pore size of the mucosalmembrane. Such absorption enhancers include sodium deoxycholate, sodiumglycocholate, dimethyl-beta-cyclodextrin,lauroyl-1-lysophosphatidylcholine and other substances having structuralsimilarities to the phospholipid domains of the mucosal membrane.

The composition can also contain other pharmaceutically acceptableexcipients for providing desirable pharmaceutical or pharmacodynamicproperties, including for example modifying or maintaining the pH,osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution of the formulation, modifying or maintaining release orabsorption into an the human or animal organism. For example, polymerssuch as polyethylene glycol may be used to obtain desirable propertiesof solubility, stability, half-life and other pharmaceuticallyadvantageous properties (Davis et al., 1978, Enzyme Eng. 4, 169-173;Burnham et al., 1994, Am. J. Hosp. Pharm. 51, 210-218). Representativeexamples of stabilizing components include polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Otherstabilizing components especially suitable in plasmid-based compositionsinclude hyaluronidase (which is thought to destabilize the extracellular matrix of the host cells as described in WO 98/53853),chloroquine, protic compounds such as propylene glycol, polyethyleneglycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or derivativesthereof, aprotic compounds such as dimethylsulfoxide (DMSO),diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane,dimethyl-formamide, dimethylacetamide, tetramethylurea, acetonitrile(see EP 890 362), nuclease inhibitors such as actin G (WO 99/56784) andcationic salts such as magnesium (Mg²⁺) (EP 998 945) and lithium (Li⁺)(WO 01/47563) and any of their derivatives. The amount of cationic saltin the composition of the disclosure typically ranges from about 0.1 mMto about 100 mM, and still more typically from about 0.1 mM to about 10mM. Viscosity enhancing agents include sodium carboxymethylcellulose,sorbitol, and dextran. The composition can also contain substances knownin the art to promote penetration or transport across the blood barrieror membrane of a particular organ (e.g. antibody to transferrinreceptor; Friden et al., 1993, Science 259, 373-377). A gel complex ofpoly-lysine and lactose (Midoux et al., 1993, Nucleic Acid Res. 21,871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517) can beused to facilitate administration in arterial cells.

The composition of the disclosure may also comprise one or moreadjuvant(s) suitable for systemic or mucosal application in humans.Representative examples of useful adjuvants include without limitationalum, mineral oil emulsion such as Freunds complete and incomplete,lipopolysaccharide or a derivative thereof (Ribi et al., 1986,Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ.Corp., NY, p407-419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72, 4931-4939; WO 98/56415), Escin, Digitonin, Gypsophila orChenopodium quinoa saponins and CpG oligodeoxynucleotides. Alternativelythe composition of the disclosure may be formulated with conventionalvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, and lipid-based particles, etc.The composition may also be formulated in the presence of cholesterol toform particulate structures such as liposomes.

The composition may be administered to patients in an amount effective,especially to enhance an immune response in an animal or human organism.As used herein, the term “effective amount” refers to an amountsufficient to realize a desired biological effect. For example, aneffective amount for enhancing an immune response could be that amountnecessary to cause activation of the immune system.

The appropriate dosage may vary depending upon known factors such as thepharmacodynamic characteristics of the particular active agent, age,health, and weight of the host organism; the condition(s) to be treated,nature and extent of symptoms, kind of concurrent treatment, frequencyof treatment, the need for prevention or therapy and/or the effectdesired. The dosage will also be calculated dependent upon theparticular route of administration selected. Further refinement of thecalculations necessary to determine the appropriate dosage for treatmentis routinely made by a practitioner, in the light of the relevantcircumstances. The titer may be determined by conventional techniques. Acomposition based on vector plasmids may be formulated in the form ofdoses of between 1 □g to 100 mg, advantageously between 10 □g and 10 mgand typically between 100 □g and 1 mg. A composition based on proteinsmay be formulated in the form of doses of between 10 ng to 100 mg. Atypical dose is from about 1 □g to about 10 mg of the therapeuticprotein per kg body weight. The administration may take place in asingle dose or a dose repeated one or several times after a certain timeinterval. In one typical embodiment, the composition of the presentdisclosure is administered by injection using conventional syringes andneedles, or devices designed for ballistic delivery of solidcompositions (WO 99/27961), or needleless pressure liquid jet device(U.S. Pat. No. 4,596,556; U.S. Pat. No. 5,993,412).

The composition of the disclosure can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic. Inall cases, the composition must be sterile and should be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Sterile injectable solutions can be prepared by incorporating the activeagent (e.g., a fusion protein or infectious particles) in the requiredamount with one or a combination of ingredients enumerated above,followed by filtered sterilization.

Methods of Use

In certain embodiments, the disclosure relates to methods of vaccinationcomprising administering an effective amount of the chimeric virus-likeparticle disclosed herein to a subject in need thereof. In certainembodiments, the disclosure relates to methods of treating or preventinga viral infection comprising administering an effective amount of thechimeric virus-like particle as disclosed herein to a subject in needthereof. In certain embodiments, the administration is intramuscular. Incertain embodiments, a second administration is provided more than twoweeks after an initial administration. In certain embodiments, thesecond administration is intranasal. In certain embodiments, the methodsfurther comprise a third administration. In certain embodiments, themethods further comprise administration is in combination with theadministration of another antiviral agent, antigen, adjuvant, orvaccine.

Other pathologic diseases and conditions are also contemplated in thecontext of the disclosure, especially infectious diseases associatedwith an infection by a pathogen such as fungi, bacteria, protozoa andviruses. Representative examples of viral pathogens include withoutlimitation human immunodeficiency virus (e.g. HIV-1 or HIV-2), humanherpes viruses (e.g. HSV1 or HSV2), cytomegalovirus, Rotavirus, EpsteinBarr virus (EBV), hepatitis virus (e.g. hepatitis B virus, hepatitis Avirus, hepatitis C virus and hepatitis E virus), varicella-zoster virus(VZV), paramyxoviruses, coronaviruses; respiratory syncytial virus,parainfluenza virus, measles virus, mumps virus, flaviviruses (e.g.Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus), influenza virus, and typically humanpapilloma viruses (e.g. HPV-6, 11, 16, 18, 31. 33).

Moreover, as mentioned above, the chimeric virus-like particle, fusionprotein, nucleic acid molecule, vector, infectious particle, host celland/or composition of the present disclosure can be used as an adjuvantto enhance the immune response of an animal or human organism to aparticular antigen. This particular use of the present disclosure may bemade in combination with one or more transgenes or transgene products asdefined above, e.g. for purposes of immunotherapy. Typically, the activeagent (e.g. fusion protein, infectious particle or pharmaceuticalcomposition of the disclosure) is administered in combination with oneor more transgenes or transgene products. Accordingly, there istypically also provided a composition comprising in combination atransgene product (e.g. a viral antigen or a suicide gene product) and afusion protein as well as a composition comprising vector(s) or viralparticles encoding a transgene product and a fusion protein. Thetransgene and the fusion-encoding nucleic acid sequences may beexpressed from the same vector or from separate vectors which may havethe same origin (e.g. adenoviral vectors) or a different origin (e.g. aMVA vector encoding the particular antigen and an adenoviral vectorencoding the fusion protein). The fusion protein and the transgeneproduct (or their respective encoding vectors) can be introduced intothe host cell or organism either concomitantly or sequentially eithervia the mucosal and/or systemic route.

Combination Therapies

In some embodiments, the disclosure relates to treating or preventing aviral infection by administering a chimeric virus-like particle incombination with a second antiviral agent. In further embodiments, achimeric virus-like particle is administered in combination with one ormore of the following agents: abacavir, acyclovir, acyclovir, adefovir,amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla,boceprevir, cidofovir, combivir,darunavir, delavirdine, didanosine,docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir (Tamiflu), peginterferonalfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin,raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir,stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine,trizivir, tromantadine, truvada, valaciclovir (Valtrex), valganciclovir,vicriviroc, vidarabine, viramidine zalcitabine, zanamivir (Relenza),and/or zidovudine (AZT).

Antiviral agents include, but are not limited to, protease inhibitors(PIs), integrase inhibitors, entry inhibitors (fusion inhibitors),maturation inhibitors, and reverse transcriptase inhibitors(anti-retrovirals). Combinations of antiviral agents create multipleobstacles to viral replication, i.e., to keep the number of offspringlow and reduce the possibility of a superior mutation. If a mutationthat conveys resistance to one of the agents being taken arises, theother agents continue to suppress reproduction of that mutation. Forexample, a single anti-retroviral agent has not been demonstrated tosuppress an HIV infection for long. These agents are typically taken incombinations in order to have a lasting effect. As a result, thestandard of care is to use combinations of anti-retrovirals.

Reverse transcribing viruses replicate using reverse transcription,i.e., the formation of DNA from an RNA template. Retroviruses oftenintegrate the DNA produced by reverse transcription into the hostgenome. They are susceptible to antiviral drugs that inhibit the reversetranscriptase enzyme. In certain embodiments the disclosure relates tomethods of treating viral infections by administering a chimericvirus-like particle, and a retroviral agent such as nucleoside andnucleotide reverse transcriptase inhibitors (NRTI) and/or anon-nucleoside reverse transcriptase inhibitors (NNRTI). Examples ofnucleoside reverse transcriptase inhibitors include zidovudine,didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine,entecavir, apricitabine. Examples of nucleotide reverse transcriptaseinhibitors include tenofovir and adefovir. Examples of non-nucleosidereverse transcriptase inhibitors include efavirenz, nevirapine,delavirdine, and etravirine.

In certain embodiments, the disclosure relates to methods of treating aviral infection by administering a chimeric virus-like particleoptionally with an antigen in combination with an antiviral drug, e.g.,2′,3′-dideoxyinosine and a cytostatic agent, e.g., hydroxyurea.

Human immunoglobulin G (IgG) antibodies are believed to have opsonizingand neutralizing effects against certain viruses. IgG is sometimesadministered to a subject diagnosed with immune thrombocytopenic purpura(ITP) secondary to a viral infection since certain viruses such as, HIVand hepatitis, cause ITP. In certain embodiments, the disclosure relatesto methods of treating or preventing viral infections comprisingadministering a chimeric virus-like particle in combination with animmunoglobulin to a subject. IgG is typically manufactured from largepools of human plasma that are screened to reduce the risk of undesiredvirus transmission. The Fc and Fab functions of the IgG molecule areusually retained. Therapeutic IgGs include Privigen, Hizentra, andWinRho. WinRho is an immunoglobulin (IgG) fraction containing antibodiesto the Rho(D) antigen (D antigen). The antibodies have been shown toincrease platelet counts in Rho(D) positive subjects with ITP. Themechanism is thought to be due to the formation of anti-Rho(D)(anti-D)-coated RBC complexes resulting in Fc receptor blockade, thussparing antibody-coated platelets.

EXPERIMENTAL Construction and Expression of GPI-Anchored GIFT4

To generate a gene encoding the membrane-anchored GIFT4, the codingsequences of the signal peptide from the honeybee melittin and murineCD59 GPI anchor were fused to the 5′- and 3′-ends of the GIFT4 codinggene (derived from mouse sequences) in frame to obtain the full-lengthencoding gene of a GPI-anchored GIFT4 (GPI-GIFT4) by overlapping PCR.The resulting GPI-GIFT4 encoding gene was then cloned into transfervector pFastBac-1 plasmid (Invitrogen, Carlsbad, Calif.). A recombinantbaculovirus (rBV) expressing GPI-GIFT4 was generated by using theBac-to-Bac insect cell protein expression system (Invitrogen, Carlsbad,Calif.).

To confirm whether GPI-anchored GIFT4 can be membrane-orientedtranslocated and expressed on cell surfaces, sf9 cells were infectedwith rBVs expressing GPI-GIFT4 at a MOI of 2. Two days later, cells wereharvested and stained with rat anti-mouse GM-CSF antibodies (BDBiosciences) followed by PE-conjugated secondary antibodies. Anon-GIFT4-related rat anti-mouse antibody was served as an antibodycontrol. Sf9 cells infected with rBVs expressing Env were stained withanti-GM-CSF followed by PE-conjugated secondary antibodies as anothercontrol. Fluorescent intensity was recorded and analyzed by FACS with aBD FACS Canto II flow cytometer.

Production of HIV VLPs

Four different VLPs (Gag only VLPs, GIFT4/Gag VLPs, sVLPs and cVLPs)were produced for comparison using an insect cell expression system asdescribed in Wang et al. J Virology, 2007, 81, 10869-10878. For theproduction of cVLPs, sf9 cells were co-infected with three rBVsrespectively expressing a modified HIV Env consensus (ConS) which showeda high level of incorporation into VLPs, GPI-GIFT4, and Gag, at MOIs of6, 2 and 3, respectively. Standard VLPs and Gag only VLPs were alsoproduced. GIFT4/Gag VLPs were produced by co-infection of sf9 cells withrBVs expressing GPI-GIFT4 and Gag at MOIs of 2 and 3, respectively. Twodays post-infection, the culture supernatant was collected and VLPs wereconcentrated by porous fiber filtration using the Quixstand benchtopsystem (GE Healthcare, Uppsala, Sweden) followed by sucrose densitygradient ultracentrifugation. To quantitate the yield of purified VLPs,the protein concentration of each sample was estimated using the Bio-Radprotein assay (Bio-Rad Laboratories, Inc, Hercules, Calif.).

A diagram of the membrane-bound form of GIFT4 gene is shown in FIG. 1A.The melittin signal peptide and CD59 GPI anchoring signal-codingsequences were fused to 5′ and 3′-ends of the GIFT4 encoding sequence inframe to facilitate the membrane insertion of GPI-GIFT4. Western blot(FIG. 1B) using anti-GM-CSF antibody detected a band migrating at 37 kDain the lysate of sf9 cells infected by recombinant baculovirus (rBVs)expressing the GPI-GIFT4 gene (lane 1 in FIG. 1B), corresponding to theexpected size of GPI-anchored GIFT4. The membrane anchoring of theexpressed GPI-GIFT4 was further demonstrated by the enhanced fluorescentintensity measured by FACS analysis of the rBV-infected cells after cellsurface staining with anti-GM-CSF antibodies, followed by PE-conjugatedsecondary antibodies.

VLPs were produced using the rBV expression system in insect cells. Theprotein composition of the resulting VLPs was characterized by westernblot using antibodies specific to Gag, Env or GM-CSF. As shown in FIG. 2(a.2), the Env incorporated into standard Env/Gag VLPs (sVLPs, lane 2)or cVLPs (lane 4) was observed to have a molecular mass of about 120kDa. A band migrating at the expected size of GPI-GIFT4 was seen in cVLPand GIFT4/Gag VLPs (lanes 3 and 4 in a.3), demonstrating theincorporation of GPI-GIFT4 into these VLPs. The results shown in lane 4of both a.2 and a.3 in FIG. 2 further indicate that membrane-anchoredGIFT4 and Env can be co-incorporated into HIV cVLPs. To verify that theintegration of GIFT4 into VLPs is through GPI anchoring on the membranesurface, FACS assay was carried out. GIFT4 was detected by the enhancedfluorescent intensity in cVLPs but not sVLPs after anti-GM-CSF antibodystaining. Further, the GIFT4 signal from cVLPs was completely eliminatedby treatment with PIPLC, a phosphatidylinositol-specific phospholipasewhich releases GPI-anchored molecules from membranes, as shown in FIG.2b . Together, these data demonstrated that GIFT4 can be incorporatedinto VLPs, or co-incorporated into cVLPs, through GPI anchoring.

Functional Characterization of GIFT4-Containing VLPs

To determine whether the anchored GIFT4 in cVLPs retains the biologicalactivity of soluble GIFT4, whether cVLPs can induce proliferation ofguinea pigs spleen cells in vitro were tested. The spleen cells werecultured in complete RPMI medium in the presence of 1μg/ml sVLPs, cVLPs,and GIFT4/Gag VLPs, respectively. Soluble GIFT4 (50 ng/ml) was used as apositive control. Following incubation at 37° C. in 5% CO2 for 2 days,the proliferation of cells was observed and imaged under an EVOSmicroscope (Life Technologies, Grand Island, N.Y.).

After culturing for 2 days in the presence of 1 μg/ml of cVLPs orGIFT4/Gag VLPs, was significantly higher numbers of spleen cellsproliferated into colonies with larger colony sizes when compared to thecontrol or sVLPs. Proliferation was also observed in sVLP-treated cells,although at a lower level when compared to that of the GIFT4 containingVLPs, demonstrating that VLPs themselves are also lymphocytestimulators. These results indicate that GPI-GIFT4 incorporated intoVLPs retains the biological activity of the soluble GIFT4 in stimulationof lymphocyte proliferation.

Enhanced Systemic Antibody Responses to cVLPs

To investigate whether GIFT4 incorporated into HIV VLPs enhancesantibody responses against the Env immunogen, groups of guinea pigs wereimmunized with one intramuscular (i.m.) prime followed by two intranasal(i.n.) boosts with sVLPs, cVLPs, GIFT4/Gag VLPs or Gag only VLPs,respectively. Immune serum IgG levels specific to HIV Env at 2 weeksafter each immunization were assessed by ELISA. The results shown inFIG. 3A (presented as endpoint titers) demonstrate that cVLPs inducedserum antibody responses with higher titers than those observed withsVLPs (P<0.05). After three immunizations (bleed 3 at week 10, FIG. 3a), guinea pigs immunized with cVLPs exhibited 5-fold higher IgG levelsthan those induced by sVLPs (means of 24600 vs. 4666, P<0.01). Theseresults indicated that co-incorporation of the membrane-anchored GIFT4into VLPs is highly effective in enhancing anti-Env immune responses.Although cVLPs induced elevated IgG responses, Env-specific IgA inimmune sera was not detected.

The serum IgG subclass profiles were assessed in the bleed 3 sera, andit was observed that sVLPs and cVLPs induced both IgG1 and IgG2 immuneresponses. The IgG2/IgG1 ratio for the sVLP group is about 9, and about7 for the cVLP group (FIGS. 3b and 3c ). Based on these data, IgG2dominates the IgG responses to HIV VLPs. Although cVLPs induced higherIgG titers compared to sVLPs, their antibody responses show similar IgGsubtype profiles. The sequence of GIFT4 used was derived from mice. Thuswhether antibody responses specific to GIFT4 were induced in guineapigs, and whether these antibodies decrease the adjuvant function ofGIFT4 in subsequent immunizations as invesitgated. GIFT4-specificantibodies in immune sera was not observed.

Enhanced Mucosal Antibody Responses to cVLPs

Female Hartley strain guinea pigs were obtained from Charles RiverLaboratory (Wilmington, Mass.) and were separated into four groups (5animals per group). Groups were immunized with an immunization regimenincluding one intramuscular (i.m.) prime followed by two intranasal(i.n.) boosts with VLP vaccines at intervals of 4 weeks. For eachimmunization, animals in the Gag only and GIFT4/Gag VLP groups wereimmunized with 100 μg total protein, respectively. Standard and cVLPswere administered using doses containing 10 μg Env, respectively. Asaverages, one dose of GIFT4-containing VLPs (cVLPs and GIFT4/Gag VLPs)contained about 2 μg GIFT4 calibrated by using soluble GIFT4. Two weeksafter each immunization, immune sera were collected by vena cavableeding of anesthetized guinea pigs.

Mucosal immunity is important for controlling a primary HIV-1 infection.To determine whether cVLPs induce enhanced mucosal immune responses bythis immunization regimen, the secretory Env-specific IgA and IgG levelsin saliva and vaginal washes were evaluated after three immunizations.As shown in FIGS. 4a and 4b , both Env-specific IgG and IgA titers insaliva samples were found to be much higher in the cVLP group than thatin the sVLP group. Remarkably, at week 10, cVLP-immunized guinea pigsalso showed about 5-fold higher IgG levels (FIGS. 4c ) and 6-fold higherIgA levels (FIG. 4d ) in vaginal washes than those induced insVLP-immunized guinea pigs, demonstrating that the GIFT4 is an effectiveadjuvant for eliciting mucosal immune responses.

Enhanced Antibody Avidity

Antibody avidity for the HIV antigen is low at the early stage ofinfection and increases as the infection progresses while antibodymatures. Neutralizing antibodies with increased avidity evolve duringmaturation. A significant increase in avidity has been reported afterrepeated antigen exposure. Several recent studies have also showncorrelations between the avidity of non-neutralizing antibodies and HIVprotective efficacy. Therefore, antibody avidity analysis is aneffective way to evaluate antibody quality for providing protection. Todetermine whether cVLPs induce antibody responses to Env with enhancedavidity, six Env-pseudotyped viruses from both clades B and C, werecompared. Because Env is inserted into the pseudoviral envelope, as isthe case in virions. Env in pseudoviruses is functionally equal to it inviruses also, binding to target cells and mediating virus-host cellmembrane fusion. Env in pseudoviruses exactly corresponds to that inviruses. Pseudotyped virus-based neutralizing assays have beenextensively used to evaluate an antibody capacity for blocking HIVinfection. Thus antibody avidity to pseudoviruses Env should reflect theantibody binding to HIV particles. The results shown in FIGS. 5A-Bdemonstrated that serum antibodies in the cVLP group showedsignificantly increased avidity compared to the sera from the sVLPimmunized group. The cVLPs elicited antibodies with increased aviditywith AIs around 40 for binding to 4 of the 6 clade B strains (FIG. 5A)as well as 4 of 6 clade C viruses (FIG. 5B) compared with sVLPs with AIsno more than 20 (P<0.05). Intermediate levels of avidity enhancementwere found to strain 6535.3 in clade B and ZM214M.PL15 in clade C, andno change was observed with AC10.0.29 in clade B or ZM109F.PB4 (clade C)(P>0.05). Interestingly, although cVLPs elicited increased avidity tosVLPs as observed above, avidity to different Env among these strainscompared are not significantly different.

Enhanced Antibody Neutralizing Breadth and Potency

Neutralizing antibodies can directly block viral infection by bindingtightly to the functional Env, mediating virus aggregation,complement-dependent inactivation, or triggering antibody-dependentcell-mediated cytotoxicity/virus inhibition (ADCC/ADCVI), and thus areideal targets to be elicited by a vaccine. Our results demonstrate thatHIV cVLPs containing GPI-anchored GIFT4 induced higher titers of IgGcompared to sVLPs. The neutralization reactivity of these antibodies wasinvestigated using a panel of HIV clade B and C Env-pseudoviruses, thesame virus panel as was used to compare antibody binding avidity inFIGS. 5A-B. As shown in FIG. 6A, serum neutralizing reactivity elicitedby the cVLP group against PVO.4, a tier 3 virus which shows strongresistance to neutralization, and RHPA4259.7 (tier 2) were enhanced(approximately 30%-40% of the viruses were neutralized to lower than 20,P<0.05) compared to the sVLP group. Of the 6 clade C viruses tested,immune sera from cVLP group exhibited enhanced neutralization toDu156.12 (tier 2), ZM214M.PL15 (tier 2) and ZM109F.PB4 (intermediate)compared to sVLP group (P<0.05) (FIG. 6B). GIFT4-containing VLP and sVLPgroups showed similar neutralization titers to the other viruses(P>0.05). These results further indicate an adjuvant effect of themembrane-anchored GIFT4 in cVLPs in inducing antibody responses withenhanced neutralizing breadth and potency.

1. A nucleic acid comprising a segment encoding a fusion proteincomprising granulocyte-macrophage colony-stimulating factor andinterleukin 4 and a glycosylphosphatidylinositol signal sequence.
 2. Thenucleic acid of claim 1 wherein the granulocyte-macrophagecolony-stimulating factor segment comprisesMWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO: 1) or variant with greater than 70%identity.
 3. The nucleic acid of claim 1 wherein the interleukin 4segment comprisesMGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS (SEQ ID NO: 2) or variant withgreater than 70% identity.
 4. The nucleic acid of claim 1 wherein theglycosylphosphatidylinositol signal sequence CD59 comprisesLSNGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 5) or variant with greaterthan 70% identity.
 5. The nucleic acid of claim 1 wherein the fusionprotein further comprises a melittin signal peptide comprisingMKFLVNVALVFMVVYISYIYA (SEQ ID NO:6) or variant with greater than 70%identity.
 6. The nucleic acid of claim 1 wherein the fusion proteinhuman MKFLVNVALVFMVVYISYIYAMWLQ SLLLLGTVAC SISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQEGGGGGMGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSSLSNGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 7), orvariant with greater than 70% identity.
 7. An isolated fusion proteinencoded by the nucleic acid of claim
 1. 8. A vector comprising thenucleic acid of claim 1 in operable combination with a promotersequence.
 9. A chimeric virus-like particle comprising the fusionprotein encoded by the nucleic of claim
 1. 10. The chimeric virus-likeparticle of claim 9, further comprising a viral envelope protein. 11.The chimeric virus-like particle of claim 10, wherein the viral envelopeprotein is and HIV envelope protein.
 12. The chimeric virus-likeparticle of claim 10, wherein the viral envelope is gp160, gp120, orgp41.
 13. A cell comprising the vector of claim 8 or the nucleic acid ofclaim 1
 14. A method of vaccination comprising administering aneffective amount of the chimeric virus-like particle of claim 9 to asubject in need thereof.
 15. The method of claim 9 wherein theadministration is intramuscular.
 16. The method of claim 14, wherein asecond administration is provided more than two weeks after an initialadministration.
 17. The method of claim 16, wherein the secondadministration is intranasal.
 18. The method of claim 17, furthercomprising a third administration.
 19. A method of treating orpreventing a viral infection comprising administering an effectiveamount of the chimeric virus-like particle of claim 9 to a subject inneed thereof.
 20. The method of claim 19, wherein the administration isin combination with the administration of antiviral agent, antigen,adjuvant, or vaccine.